Vaccinia virus growth in BSC-1 or HeLa cells was inhibited by aphidicolin concentrations greater than 20 Mum. After treatment of the wild-type virus with hydroxylamine, a viral mutant was isolated which is resistant to 80 Mum aphidicolin. In an in vitro assay viral DNA polymerase isolated from cells infected with mutant virus was more resistant to aphidicolin than viral DNA polymerase isolated from cells infected with wild-type virus. Transfer of aphidicolin-resistance was achieved by transfecting cells infected with wild-type virus with DNA isolated from aphidicolin-resistant virus. Production of aphidicolin-resistant recombinant viruses was measured by plaque assay in the presence of aphidicolin. The site of the mutation was initially located in the HindIII E segment of the resistant DNA. This segment, which was cloned in a pUC9 plasmid, was digested further with EcoRI. Marker rescue experiments with the resulting segments showed that the second largest, labeled Eco B, contained the drug-resistance mutation. The Eco B segment was cloned in pUC9 and the recombinant plasmid was digested with RsaI. Transfection experiments with the entire digest showed a detectable level of aphidicolin-resistant plaques which was about ten times that found for a background produced by transfection with wild-type DNA. To determine which particular Rsa segment contained the resistant site, the recombinant Eco B plasmid was digested with exonuclease III, and the undigested DNA was tested for marker rescue. Correlation of the extent of exonuclease digestion with the map positions of the Rsa sites indicated that a segment labeled Rsa H might contain the mutation. This segment was cloned in pUC9 and the recombinant DNA was used to transfer the aphidicolin-resistance marker. Since the active Rsa DNA segment is about 194 bp, it should be easy to sequence with the M13 phage system.